Singlet-Oxygen-Mediated Regulation of Photosynthesis-Specific Genes: A Role for Reactive Electrophiles in Signal Transduction

During photosynthesis, reactive oxygen species (ROS) are formed, including hydrogen peroxide (H2O2) and singlet oxygen (1O2), which have putative roles in signalling, but their involvement in photosynthetic acclimation is unclear. Due to extreme reactivity and a short lifetime, 1O2 signalling occurs via its reaction products, such as oxidised poly-unsaturated fatty acids in thylakoid membranes. The resulting lipid peroxides decay to various aldehydes and reactive electrophile species (RES). Here, we investigated the role of ROS in the signal transduction of high light (HL), focusing on GreenCut2 genes unique to photosynthetic organisms. Using RNA seq. data, the transcriptional responses of Chlamydomonas reinhardtii to 2 h HL were compared with responses under low light to exogenous RES (acrolein; 4-hydroxynonenal), β-cyclocitral, a β-carotene oxidation product, as well as Rose Bengal, a 1O2-producing photosensitiser, and H2O2. HL induced significant (p < 0.05) up- and down-regulation of 108 and 23 GreenCut2 genes, respectively. Of all HL up-regulated genes, over half were also up-regulated by RES, including RBCS1 (ribulose bisphosphate carboxylase small subunit), NPQ-related PSBS1 and LHCSR1. Furthermore, 96% of the genes down-regulated by HL were also down-regulated by 1O2 or RES, including CAO1 (chlorophyllide-a oxygnease), MDH2 (NADP-malate dehydrogenase) and PGM4 (phosphoglycerate mutase) for glycolysis. In comparison, only 0–4% of HL-affected GreenCut2 genes were similarly affected by H2O2 or β-cyclocitral. Overall, 1O2 plays a significant role in signalling during the initial acclimation of C. reinhardtii to HL by up-regulating photo-protection and carbon assimilation and down-regulating specific primary metabolic pathways. Our data support that this pathway involves RES.


Introduction
Solar energy drives photosynthesis, which is fundamental for sustaining almost all life on earth.Photosynthesis starts with the capture of light energy by pigments in the protein complexes of thylakoid membranes and its transfer to chlorophyll in the reaction centres of photosystems (PS) for initiating photochemistry.Photosynthetic activity requires a variety of unique processes not found in non-photosynthetic organisms (heterotrophs).In total, 597 nucleus-encoded proteins have been identified in green lineage organisms, but not, or poorly conserved, in heterotrophs, which have collectively been grouped as GreenCut2 [1].About 30% of GreenCut2 genes are of prokaryotic origin, with about half having an unknown function and most encoding proteins predicted to be localized to the chloroplast [2].Rational for inclusion in GreenCut2 was a putative protein orthologue in the green lineage eukaryotes (Viridiplantae), including the algae Chlamydomonas reinhardtii and Ostreococcus taurii, the moss Physcomitrella patens, and the vascular plant Arabidopsis thaliana, not found in heterotrophs (e.g., Pseudomonas aeruginosa, Sulfolobus solfataricus, Caenorhabditis elegans, Homo sapiens, etc.) [1].
Molecular oxygen, a product of oxygenic photosynthesis, can form unstable intermediates called reactive oxygen species (ROS).It is well established that across the diversity of life, the many signalling networks functioning in development, or sensing physiological state and environmental change, integrate redox components involving ROS [3,4].Organelles, including mitochondria, chloroplasts and peroxisomes, are major sites of ROS production in plant cells, which can invoke responses not only locally, but also in the nucleus by activating or suppressing transcription.Here, it is important to distinguish the species of ROS, since they can have significantly different chemical properties.For example, hydrogen peroxide (H 2 O 2 ) signalling filters through catalytic sites of peroxiredoxins, thioredoxins and glutaredoxins to trigger target thiol/disulfide redox 'switches' [5].In contrast, singlet oxygen ( 1 O 2 ) is short-lived, and the associated signalling is largely in response to the oxidation of, for example, carotenoids (e.g., β-carotene), proteins (e.g., EXECUTER1) or cellular components (e.g., membranes) [6].Increases in H 2 O 2 and 1 O 2 production of C. reinhardtii have been observed in response to a 5-fold increase in light intensity [7,8], and could be involved in acclimation to high light (HL) by affecting the transcription of GreenCut2 genes.
In response to HL, photosynthetic organisms activate non-photochemical quenching (NPQ), which can safely dissipate excess light energy to heat [9].However, NPQ has limits and ROS formation is an unavoidable part of photosynthesis.In particular, 1 O 2 , formed by charge recombination in PSII, is considered the most damaging ROS produced under excess light [10,11].It has become apparent that 1 O 2 likely has a role in HL signal transduction [6,[12][13][14][15][16][17].One of the most important components of thylakoid membranes that house photosynthetic complexes are galactolipids, of which a significant fraction is composed of the poly-unsaturated fatty acid (PUFA) linolenic acid [18].The three electronrich C=C bonds of linolenic acid are prone to non-enzymatic oxidation by 1 O 2 , thereby forming lipid peroxides [19].These can spontaneously dissociate, releasing a variety of carbonyls, including short-chain α,β-unsaturated aldehydes called reactive electrophile species (RES), including acrolein and 4-hydroxynonenal (HNE), which may play a role in HL signalling.
Chlamydomonas reinhardtii was the first Chlorophyte (green alga) to have its genome sequenced [20] and has since become a model organism for investigating HL stress acclimation [17].In the model angiosperm Arabidopsis thaliana, β-cyclocitral from the oxidation of β-carotene has emerged as part of the 1 O 2 stress response via the zinc finger protein methylene blue-sensitive 1 (MBS1) protein [21].However, despite possessing an MBS1 signalling pathway [22], β-cyclocitral has a minor role in 1 O 2 signalling in C. reinhardtii [23].In contrast, the exogenous treatment of low light (LL)-treated C. reinhardtii with the RES acrolein was able to lead to a remarkably similar differential expression of genes (DEG) compared with the response to the photosensitizer Rose Bengal, which can be used to mimic HL-induced 1 O 2 production [15].
Here, using an RNA seq.approach with C. reinhardtii, we investigated the involvement of various ROS-mediated signal-transduction pathways in the HL acclimation of photosynthesis by focusing specifically on the GreenCut2 gene cluster.Comparisons were drawn between the influence of HL and cells treated under LL with H 2 O 2 or Rose Bengal, the latter of which induced a similar level of lipid peroxidation as occurred under the HL treatment [24].To investigate potential intermediates in ROS signalling, the response of cells under LL to acrolein, HNE and β-cyclocitral were included.
HL was the only treatment assessed that induced more up-than down-regulation of GreenCut2 genes (Table 2).More total GreenCut2 genes were up-regulated by Rose Bengal (n = 140) and acrolein (n = 138), but an even greater number were also down-regulated (n = 180 and n = 114, respectively, Table 2).β-cyclocitral and H 2 O 2 (from [26]) led to an up-regulation of 2 and 32 GreenCut2 genes, respectively, but <4% overlapped with the response to HL (Table 2).

Shared Genes
When considering significantly affected genes at p < 0.05 without FDR, the number of up-and down-regulated genes in response to 2 h HL increased to 108 and 23, respectively (Table 2).Many of these were also similarly regulated by exogenous treatment with Rose Bengal, or the RES acrolein and HNE (Table 2 and Table S1).
A significant positive correlation (r 2 = 0.32) was found when correlating the log2FC values of these treatments with HL for all 98 shared significantly affected genes (Figure 1), whereas for H 2 O 2 , the overall relationship with HL was negative (Figure S1), although only 16 genes were shared.However, it was also obvious that a proportion of genes up-regulated by HL was down-regulated by RES or Rose Bengal (Figure 1).We could not find a KEGG pathway represented by more than two genes within these 29 genes (Table S4).A very close relationship in the regulation of significantly affected GreenCut2 genes was found when correlating log2FC values in response to Rose Bengal and either acrolein (r 2 = 0.67; 179 genes) or HNE (r 2 = 0.78; 108 genes) or H 2 O 2 (r 2 = 0.60; 46 genes), and in response to HNE and either acrolein (r 2 = 0.71; 104 genes) or H 2 O 2 (r 2 = 0.62; 28 genes) (Figure S1).

Shared Genes
When considering significantly affected genes at p < 0.05 without FDR, the number of up-and down-regulated genes in response to 2 h HL increased to 108 and 23, respectively (Table 2).Many of these were also similarly regulated by exogenous treatment with Rose Bengal, or the RES acrolein and HNE (Tables 2 and S1).

A Role for 1 O2 but Not H2O2 in Affecting the Gene Expression of GreenCut2 under HL
To investigate how ROS contribute to the signalling of photosynthesis-specific genes during HL acclimation, we compared the differential expression of GreenCut2 genes in response to 2 h HL with 2 h treatment in response to exogenous chemicals related to ROS under LL.Previously, physiological as well as molecular responses showed that exogenous molecules (e.g., H2O2, RES and β-cyclocitral) at the concentrations investigated here were able to penetrate cells to induce a response, thus be available to act in signalling pathways [8,23,26].For example, using fluorescent H2O2 sensors in C. reinhardtii, it was shown that 0.1-1.0 mM exogenous H2O2, and chloroplast-derived H2O2 under HL leaked

A Role for 1 O 2 but Not H 2 O 2 in Affecting the Gene Expression of GreenCut2 under HL
To investigate how ROS contribute to the signalling of photosynthesis-specific genes during HL acclimation, we compared the differential expression of GreenCut2 genes in response to 2 h HL with 2 h treatment in response to exogenous chemicals related to ROS under LL.Previously, physiological as well as molecular responses showed that exogenous molecules (e.g., H 2 O 2 , RES and β-cyclocitral) at the concentrations investigated here were able to penetrate cells to induce a response, thus be available to act in signalling pathways [8,23,26].For example, using fluorescent H 2 O 2 sensors in C. reinhardtii, it was shown that 0.1-1.0 mM exogenous H 2 O 2 , and chloroplast-derived H 2 O 2 under HL leaked into the cytosol [27].Transcripts encoding the proteins involved in photosynthesis showed a general downward trend after treatment with 1 mM H 2 O 2 , but mostly insignificantly [26].By far, the most up-regulated H 2 O 2 -responsive gene was a heat shock protein, HSP22A [26], which, although also highly up-regulated by Rose Bengal, acrolein and HNE, was not up-regulated in response to HL (Table S1).This is less surprising than could be expected considering that the concentrations of H 2 O 2 in HL-treated C. reinhardtii were measured at low µM [8], similar to measurements in leaves [28] and far from the 1 mM concentration used for the RNA seq.[26].In contrast, the 1 O 2 gene marker, SOUL2 [14], was significantly up-regulated by HL (Table 1) and Rose Bengal (Table S1), supporting that 1 O 2 was contributing to signalling under the HL treatment.Moreover, of all GreenCut2 genes up-and down-regulated by HL, only 3% and 4%, respectively, were similarly regulated by H 2 O 2 , whereas 24% and 78%, respectively, were similarly regulated by Rose Bengal (Table 2).Worth mentioning is that the differential expression of GreenCut2 genes in response to 1 mM H 2 O 2 was ca.60% shared with the response to Rose Bengal (Table 1), with the log2FC gene expression of these treatments significantly correlating (Figure S1).Therefore, it seems that a high proportion of H 2 O 2 signalling under such high H 2 O 2 concentrations may pass through the same pathway(s) as 1 O 2 .Since each ROS has distinct chemical properties, this may indicate an involvement of a common oxidative modification in response to both treatments, such as lipid oxidation and derived RES.Of all measured RES, only HNE significantly accumulated in response to 1 mM H 2 O 2 (personal observation).However, we concluded that 1 O 2 has much more influence on the expression of photosynthesis-specific genes during HL acclimation than H 2 O 2 .

Intermediates of 1 O 2 Signalling Likely Include RES
The known targets of 1 O 2 in C. reinhardtii include β-carotene and PUFA, from which oxidation products could be secondary messengers in 1 O 2 signalling.It was shown by Roach et al. [23] that β-cyclocitral at concentrations used for the treatment (600 ppm same as used for acrolein) were also affecting the bioenergetics of the chloroplast and were thus entering the cells and available to act in chloroplast-to-nucleus signalling.However, like H 2 O 2 , the shared DEG in response to β-cyclocitral and HL was very limited (Figure 2; Table 2), thus unlikely to play a major role in HL signalling in C. reinhardtii.Furthermore, while β-cyclocitral down-regulated porphyrin and chlorophyll synthesis [23], HL upregulated this pathway (Table 1).As for H 2 O 2 , the concentrations of potential 1 O 2 -derived molecules formed under HL are also relevant for signalling.In HL-treated photoautotrophic C. reinhardtii, the concentration of HNE was about half that of β-cyclocitral, whereas acrolein concentrations were >5 times higher than β-cyclocitral [29].In our study, we showed that HNE or acrolein can be mediators in 1 O 2 signalling of GreenCut2 genes, as revealed by the strong correlation of the log2FC of gene expression in response to these RES and Rose Bengal (Figure S1), but due to the higher concentration of acrolein in HL-treated C. reinhardtii, we expect this RES to be more involved.
There are multiple ways 1 O 2 signal transduction can occur.The cytosolic phosphoprotein Sak1 [14], the zinc finger protein MBS [22], the PSII subunit P-2 (PsbP2) [30] and the ERE-containing bZIP transcription factor Sor1 [31] have all previously been shown to contribute in C. reinhardtii.Therefore, 1 O 2 signalling may pass through several of these pathways.RES-mediated 1 O 2 signalling was first reported in the biotic stress response of plants to pathogens [32,33].The 1 O 2 -induced oxidation of thylakoid membranes [11] and derived RES was shown in C. reinhardtii to pass through the Sor1 transcription factor [24,31].Due to the strong positive correlation of GreenCut2 genes in response to Rose Bengal and HNE or acrolein (Figure S1), and all three treatments with HL (Figure 1), RES likely transmit the 1 O 2 signal from the chloroplast to the nucleus, triggering changes in gene expression under HL.

Up-Regulation via 1 O 2 of RuBisCO Activity and Down-Regulation of Glycolysis under HL
Light intensity directly impacts photosynthetic activity and, as can be expected, upregulated many GreenCut2 genes.Those also up-regulated by HNE, acrolein and Rose Bengal include Cre02.g120100,encoding RBCS1 (ribulose-1,5-bisphosphate carboxylase [RuBisC] small subunit 1).Although this small subunit is not catalytic, it is essential for maximal RuBisCO activity [34].Also upregulated by HL and Rose Bengal was Cre17.g718950,encoding RCA2 a RuBisCO activase-like protein.Increasing CO 2 assimilation would enhance use of HL for photosynthesis, as well as preventing ROS formation from decreasing excess light absorption.Lowered RuBisCO activity in C. reinhardtii mutants led to increased ROS production under HL [35], and our results support a role for 1 O 2 -derived RES in the signalling of increasing RuBisCO activity under HL.
Of all GreenCut2 genes down-regulated by HL, 78% were also down-regulated by RB, indicating a high level of the involvement of 1 O 2 signalling in lowering the transcription of unbeneficial photosynthetic processes under HL.This group includes Cre05.g232550 and Cre06.g2723,encoding PGM4 (phosphoglycerate mutase 4) and another putative phosphoglycerate mutase, respectively, which can be involved in glycolysis by catabolising 3-phosphoglycerate (PGA), also an intermediate in the Calvin-Benson cycle.Thus, lowered PGM4 activity would increase PGA availability for inorganic carbon assimilation via RuBisCO.The involvement of 1 O 2 signalling in this pathway seems independent of RES (Table S1).However, Cre10.g466500 encoding for glyoxylase I family protein was highly down-regulated by HL, as well as by HNE, acrolein and Rose Bengal.The glyoxylase pathway breaks down the products of glycolysis.Also highly down-regulated by HL, Rose Bengal and HNE was Cre09.g410700encoding MDH5 (chloroplastic NADP-dependent malate dehydrogenase) that converts malate to oxaloacetate.MDH5 is an oxidoreductase with NADP as a ligand and is exclusively located in chloroplasts [36].Since MDH activity consumes NADPH [36], more NADPH would potentially be available for RuBisCO activity.Overall, the data indicate that 1 O 2 signalling is involved in enhancing inorganic carbon assimilation under HL.
A typical response to HL is an increase in chlorophyll a:b ratio due to less need for chlorophyll b-rich light-harvesting complexes (LHC).The gene Cre01.g043350encoding CAO1 (chlorophyllide a oxygenase), which plays a role in chlorophyll b synthesis, was down-regulated by HL, RB, HNE and acrolein, supporting a role for 1 O 2 -derived RES in this response.

Photoprotection Was Up-Regulated by Acrolein Without HL
The dissipation of excess light energy to heat via NPQ is a universal strategy of photosynthetic organisms activated by HL [9].In C. reinahardtii, NPQ via LHC-stressrelated (LHCSR) proteins protects from 1 O 2 formation and photoinhibition [23,29].Of the 15 GreenCut2 genes most up-regulated by HL, 10 were also significantly up-regulated by acrolein, despite acrolein treatments being made under LL (Table 2).Included in this group are Cre01.g016750encoding a PSBS protein, Cre01.g016600encoding PSBS1 (the two most up-regulated GreenCut2 genes), Cre09.g394325encoding ELI3 (Early lightinducible protein) and Cre02.g109950encoding HLIP (single-helix LHC light protein).In A. thaliana, ELIP2 (ELI3 homologue in C. reinhardtii) was shown to be involved in various stress responses, such as cold and UV-B [37], while PSBS1 is required for the activation of NPQ, possibly by promoting the conformational changes needed for the activation of LHCSR-dependent quenching in the antenna of PSII [38,39].Although not a GreenCut2 gene, LHCSR1 is also strongly up-regulated by HL and acrolein [24].Overall, acrolein seems to have an important function in the acclimation process against HL by up-regulating photoprotection.

Material Source and Origin of Data
Chlamydomonas reinhardtii wild-type (WT) strain 4A mt + (CC-4051) was used for all analyses.For treatments, axenic cultures were established in TAP (TRIS-acetate-phosphate) media under LL at 50 µmol quanta m −2 s −1 (sub-saturating), and once in exponential phase, were transferred to photoautotrophic THP media or placed on solid agar TAP media, depending on the treatment.For volatile treatments (acrolein, β-cyclocitral), homogenous algal 'lawns' were initiated on agar by distributing 0.75 mL of liquid TAP culture evenly across 11 cm Petri dishes half-filled with TAP +1.5% agar and left for 0.5 h in a laminar flow bench to evaporate the liquid media in sterile air.The lid was then closed but not sealed, and the cells were cultivated for 4 days at 20 • C under LL before treatment with volatile acrolein or β-cyclocitral (both Sigma-Aldrich, St. Louis, MI, USA).For more details, see [23,24].For treatments of liquid cultures (HL, Rose Bengal, HNE), TAP cultures were transferred to a photoautotrophic THP medium, whereby the media was pH-adjusted to 7.0 with HCl rather than with acetic acid.The cells were pelleted at 1000× g for 1 min and TAP media was replaced with THP.Liquid THP cultures were bubbled with sterile air, achieved with a 0.2 µM air-filter (Minisart NML Plus cellulose acetate filters; Sartorius, Göttingen, Germany) and cultivated for at least 24 h under LL with a culture rotation at 75 rpm before treatment.The concentration of HNE (>98% pure, Cayman Chemical Co.Ann Arbor, MI, USA) in the culture was 37.5 µM with 14.6 µL of 10 mg/mL ethanol stock solution added to 25 mL culture under LL, which was calculated to be the same concentration as for the volatile acrolein treatment [24].Cells were treated with 1 µM Rose Bengal dissolved in H 2 O (95% pure disodium salt, Sigma-Aldrich) under LL, which provides a tolerable dose of 1 O 2 [23].For HL treatment, the light intensity was increased to 750 µmol quanta m −2 s −1 (ca.double the saturating light intensity for C. reinhardtii [7]).Liquid cultures were at a density of 15 µg chlorophyll ml −1 for all samples, and during all treatments, they were rotated, but no longer bubbled with air.For the analysis of differential gene expression, comparisons were either made to the respective non-treated liquid or agar-grown cultures from LL.Three separate cultures were used as replicates for controls and treatments.
Total RNA was extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and additional on-column DNAse treatment (RNAse-free DNAse set, Qiagen) according to the manufacturer's instructions.Briefly, 15 mg of agar culture carefully scraped from the surface or 25 mg of pelleted liquid culture (after centrifugation for 1 min at 1000× g) were frozen with three 3 mm RNAse free glass beads in a 2 mL Eppendorf tube and stored at -80 • C.They were then shaken in pre-ice-cooled adaptors for 2 min at 30 Hz (TissueLyzer II, Qiagen) and 450 µL of an RLC buffer with β-mercaptoethanol was added immediately.After extraction, the samples were stored at -80 • C before the poly A enrichment of mRNA.The RNA seq.was performed by the NGS Core Facility of the Vienna Biocenter, Vienna, Austria, with Illumina's HiSeq2500 instrument using single-end sequencing with 50 bp read length.Raw reads were aligned against the C. reinhardtii reference genome (JGI v5.5 release) with STAR version 2.5.1b using a 2-pass alignment mode.Three biological replicates were analysed for each treatment and a significant FC was considered at p < 0.05 with or without FDR, as calculated with the Limma package [40].
The Algal Functional Annotation Tool was used (http://pathways.mcdb.ucla.edu/algal/index.html,accessed on 4 June 2024) for exploring the KEGG pathways of shared DEGs.

Conclusions
Previously, we have shown that 1 O 2 formation during HL peroxidises PUFA in thylakoid membranes, leading to the release of RES.Here, evidence is provided that RES, such as acrolein and HNE, can be the secondary messengers of 1 O 2 signalling, up-regulating photo-protection and possibly carbon assimilation, while down-regulating specific primary metabolic pathways towards HL acclimation.In contrast, we found that H 2 O 2 and β-cyclocitral most likely do not contribute.

Figure 1 .
Figure 1.Correlation of the expression levels of GreenCut2 genes significantly affected by high light (HL) and singlet oxygen ( 1 O2)-related treatments under low light.Data are separated along the xaxis by a log2-fold change (log2FC) in response to HL and along the y-axis by log2FC in response to exogenous treatments (acrolein; black, HNE; dark blue, Rose Bengal ( 1 O2); turquoise).The line of best fit is of all data.

Figure 1 .
Figure 1.Correlation of the expression levels of GreenCut2 genes significantly affected by high light (HL) and singlet oxygen ( 1 O 2 )-related treatments under low light.Data are separated along the x-axis by a log2-fold change (log2FC) in response to HL and along the y-axis by log2FC in response to exogenous treatments (acrolein; black, HNE; dark blue, Rose Bengal ( 1 O 2 ); turquoise).The line of best fit is of all data.

Table 1 .
GreenCut2 genes significantly affected by 2 h HL.Significance was considered by p < 0.05 with FDR and >2-fold change (FC) relative to LL conditions, i.e., >1 or <−1 after log2 transformation.Red and blue shading of FC values denotes up-and down-regulation, respectively.When known, the gene name, protein description and putative or likely function are provided.

Table 2 .
Number of differentially expressed GreenCut2 genes and the % shared amongst treatments.
Numbers below the arrows indicate total up-(↑) and down-(↓)regulated GreenCut2 genes in response to treatments listed above (p < 0.05 without FDR).Numbers in the table indicate % of the total ↑ or ↓ DEG listed on top that were also ↑ or ↓ regulated, respectively, by the treatments listed vertical-left.HL: high light, 1 O 2 : Rose Bengal, HNE: 4-hydoxynonenal, β-CC: β-cyclocitral, H 2 O 2 : hydrogen peroxide.